F-spondin (FSP) is a gene that is predominantly expressed during the early development of the vertebrate nervous system. The main function is thought to be in neural cell pattern formation and axonal growth. It was found in a subtractive hybridization screen designed to isolate floor-plate specific genes. The floor-plate provides diffusible signals that act on the neurons that extend from the developing spinal cord. These signals can lead to chemoattraction and fasciculation of commissural axons in the ventral midline. F-spondin mRNA is expressed at high levels in the developing neural tube at the ventral midline even before cell differentiation markers can detect the floor-plate. F-spondin is not detectable in other regions of the spinal cord until later in embryonic life. There is also transient F-spondin expression early in peripheral nerve development which diminishes to undetectable levels following birth. The adult central nervous system contains F-spondin while the peripheral nerve (sciatic nerve) does not. Outside the adult nervous system, organs such as the lung and kidney also express F-spondin. The protein is 807 amino acids and codes for a predicted 90 kD polypeptide. The apparent size is approximately 116 kD by SDS-PAGE which indicates post-translational modifications such as glycosylation. There are six domains homologous to the thrombospondin (TSP) type 1 repeats (TSR) which have been shown to control cell adhesion. The protein has been expressed in COS cells and purified as a myc-tag fusion protein. This protein was active in promoting neurite extension and adhesion of embryonic dorsal root ganglion and dorsal spinal cords respectively. It was not chemotropic for embryonic dorsal spinal cord neurons. (Klar, A. et al., Cell, 69:95-110 (1992)).
The C-terminal half of F-spondin contains 6 repeats identified in thrombospondin and other proteins implicated in cell adhesion. Thrombospondin is a 450,000-dalton glyco-protein secreted by platelets in response to such physiological activators as thrombin and collagen (Lawler, J., Blood, 67:1197-1209 (1986)). TSP comprises 3% of the total platelet protein and 25% of the total platelet-secreted proteins (Tuszynski, G. P., et al., J. Biol. Chem., 260:12240-12245 (1985)). Although the precise biological role of TSP has yet to be fully established, it is generally accepted that TSP plays a major role in cell adhesion and cell-cell interactions. It should be pointed out that the C-terminal repeats present in thrombospondin may have different biological activities.
TSP was found to promote the cell-substratum adhesion of a variety of cells, including platelets, melanoma cells, smooth muscle cells, endothelial cells, fibroblasts and epithelial cells (Tuszynski, G. P., et al., Science (Washington, D.C.), 236:1570-1573 (1983)).
Thrombospondin has been postulated to play a role in malarial infection induced by only one strain of malaria, plasmodium falciparum. During malarial infection, TSP promotes adhesion of parasitized red cells to endothelial cells (Roberts, D. D., et al., Nature (Lond.), 318:64-66 (1984)) and during tumor cell metastases TSP promotes adhesion of mouse sarcoma cells to the vascular bed and expression of the malignant phenotype of small cell carcinoma (Castle, V. J., J. Clin. Invest., 87:1883-1883 (1991)).
Properdin is a complement-binding protein which also contains the 6 terminal repeats found in thrombospondin. UNC-5, a C. elegans gene that bears two terminal repeats, appears to guide the axonal extension of the sub-set of neurons. These proteins, which contain at least one member of the six terminal repeats, form a family of proteins which have related functions.
The gene and polypeptide encoded thereby of the present invention has been putatively identified as an Neuronal Attachment Factor-1 protein as a result of amino acid sequence homology to rat F-spondin.